ATP-sensitive potassium channels (KATP channels) are present in many cell types, and their activity in cardiac myocytes is thought to be related to the etiology of ischemic heart disease. KATP channels sense changes in intracellular ATP concentrations and convert this signal to changes in membrane potential and membrane potassium permeability. Previous studies in this and other laboratories have shown that membrane phosphoinositides (PPIs) modulate KATPchannel activity and ATP sensitivity. Electrophysiological results suggest that these effects are due to a direct and specific interaction between PPIs and the channelforming subunit (Kir6.2) of the KATPchannel. The overall objective of this research proposal is to clarify the structural basis responsible for this specific PPI-KATP channel interaction, and to elucidate how it affects ATP sensitivity. Our recent work using biochemical binding assays shows that the ability of PPIs to bind Kir6.2 depends on the headgroup of the PPI. We have also shown that PPIs reduce photolabeling of Kir6.2 by ATP photoaffinity analogs. Based on these preliminary results, we propose to address the following Specific Aims: (1) We will investigate the selectivity of PPI binding and structural requirement in the PPI-KATP channel interaction in order to determine whether PPIs act on Kir6.2 through a direct and structure-specific interaction. (2) We will investigate the structural mechanism for ATP binding and whether PPI-Kir6.2 association modulates ATP binding. (3) We will investigate the role of SUR2, the regulatory subunit of the KATPchannel, in PPI-KATP channel interaction and PPI modulation of ATP binding. Using a variety of complementary techniques, including binding assays, photolabeling, enzymatic digestion analysis, sitedirected mutagenesis, and electrophysiological measurements, we will provide direct evidence demonstrating how PPIs and KATP channels interact, and how this interaction modulates ATP binding. The results of this work will significantly improve our understanding of how KATpchannels respond to PPI-mediated signal transduction in heart. The results will also offer insightful structural information on the ATP sensitivity of this channel. In the broader area of ion channel physiology, knowledge gained from study of the mechanisms of PPI-KATP channel interaction and use of KATPchannel function as a model system will help us to understand the general principles that dominate specific lipid-channel interactions and channel functions.